Comparative Efficacy of East Asian Herbal Formulae Containing Astragali Radix–Cinnamomi Ramulus Herb-Pair against Diabetic Peripheral Neuropathy and Mechanism Prediction: A Bayesian Network Meta-Analysis Integrated with Network Pharmacology

The Astragali Radix–Cinnamomi Ramulus herb-pair (ACP) has been widely used in the treatment of diabetic peripheral neuropathy (DPN) as part of East Asian herbal medicine (EAHM). Eligible randomized controlled trials (RCTs) were identified by searching 10 databases. The outcomes investigated were response rate, sensory nerve conduction velocity (SNCV), and motor nerve conduction velocity (MNCV) in four regions of the body. The compounds in the ACP and their targets of action, disease targets, common targets, and other relevant information were filtered using network pharmacology. Forty-eight RCTs, with 4308 participants, and 16 different interventions were identified. Significant differences were observed in the response rate, MNCV, and SNCV, as all EAHM interventions were superior to conventional medicine or lifestyle modification. The EAHM formula containing the ACP ranked highest in more than half of the assessed outcomes. Furthermore, major compounds, such as quercetin, kaempferol, isorhamnetin, formononetin, and beta-sitosterol, were found to suppress the symptoms of DPN. The results of this study suggest that EAHM may increase therapeutic efficacy in DPN management, and EAHM formulations containing the ACP may be more suitable for improving treatment response rates to NCV and DPN therapy.


Introduction
Diabetic peripheral neuropathy (DPN) is a common complication, affecting around half of diabetic patients [1,2]. Both diabetes and prediabetes can cause DPN, leading to various types of nerve damage and accompanying clinical findings [3][4][5]. Accordingly, approximately 40% of patients with DPN develop neuropathic pain that does not respond to treatment, and various motor dysfunctions and sensory losses [4,6]. Owing to these pathophysiological characteristics, DPN not only reduces the quality of life of patients but also imposes an immense social burden. A recent study reinforced this problem by reporting that the medical expenses of patients with painful DPN are 20% higher than those of diabetic patients without corresponding complications, and the cost increases over time [7]. Various interventions for the treatment and management of DPN have been discussed; however, no disease-modifying treatment is available [1,8]. Currently, the main therapies focus on symptomatic pain relief using conventional medicines (CM), such as anticonvulsants, antidepressants, methylcobalamin, and alpha-lipoic acid [9,10]. Therefore,

Type of Studies
Only randomized controlled trials (RCTs) evaluating the efficacy and safety of oral administration of EAHM for DPN were included. There were no restrictions on language, publication date, or type of diabetes. Studies were excluded if they met any of the following criteria: (a) not an RCT or quasi-RCT; (b) a control group was not used or was inappropriate; (c) unrelated to manifestations due to DPN; (d) animal experiments; (e) case reports or reviews; or (f) not published in peer-reviewed scientific journals, including postgraduate theses or dissertations.

Type of Participants
Trials were considered eligible for inclusion if they were conducted on adults (age > 18 years) diagnosed with DPN with no restrictions on age, sex, or race.

Type of Interventions
RCTs trials comparing EAHM as an active intervention in the treatment group with CM in the control group were included. However, RCTs that used a combination of EAHM and CM as an intervention were beyond the scope of this review and were omitted. All dosage forms of EAHM intervention for symptom management in DPN, such as decoctions, granules, and capsules, were included. Studies in which East Asian medical interventions such as acupuncture, massage, or other nondrug therapies were only combined in the treatment group were excluded. Studies in which the control group included other EAHM were excluded. Even if all other inclusion criteria were satisfied, RCTs in which the exact constituent herbs of the EAHM formulation used as an intervention were not identified were excluded.

Type of Outcome Measures
The remission rate of DPN-related global symptoms observed according to the explicit criteria was selected as the outcome measure. However, most of the included studies reported the remission rates of complete remission (CR), partial remission (PR), mild remission (MR), and no remission (NR) as CR + PR/all patients. Considering that the remission rates reported by individual studies would have led to inconsistencies in the outcomes because different studies used different categorization criteria, the proportion of patients who achieved symptom alleviation in each group was used as the response rate in this review, and various study results were converted into this system.
The first set of secondary outcomes was indices evaluating motor nerve conduction velocities associated with neurological abnormalities in patients with DPN. Therefore, A pairwise meta-analysis (PMA) was performed to directly compare the EAHM with the comparator. Evidence synthesis of the included studies using the available data was performed by calculating the effect size and 95% confidence interval (CI) using a randomeffects model. Heterogeneity was considered statistically significant when the p-value based on the χ 2 test was <0.10 or I 2 was ≥50%. Two-sided p < 0.05 was considered statistically significant. Statistical synthesis of individual research results was performed using the software R (version 4.1.2; R Foundation for Statistical Computing, Vienna, Austria) and RStudio version 2022.02.3 build 492 (Integrated Development for R. RStudio, PBC, Boston, MA, USA) using the default settings of the "meta" and "metafor" package [47]. The RR and 95% confidence interval (CI) were calculated for the response rate. The mean difference (MD) and 95% confidence interval (CI) were calculated for the motor and sensory nerve conduction velocities. If heterogeneity was observed in the synthesized meta-analysis results for outcome measures involving > 10 trials, the cause of heterogeneity was traced using sensitivity analysis. To distinguish publication bias, a contour-enhanced funnel plot that included most of the studies was used [48]. To address the asymmetry of the visually confirmed funnel plot, Egger's test [49] and Begg's test [50] were performed to confirm publication bias. The overall quality of evidence for each outcome was evaluated using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) Pro [51]. The GRADE assessment evaluated the overall quality of evidence on four levels: very low, low, moderate, and high. The level of evidence is lowered by factors such as the risk of bias, inconsistency, indirectness, imprecision, and publication bias.

Network Meta-Analysis
A network meta-analysis (NMA) was performed to evaluate the relative efficacy of EAHM formulae containing AC herb-pairs and other interventions. In this review, Bayesian NMAs were performed using R v. 4.1.2, and RStudio V. 2022.02.3 build 492 to evaluate the comparative effectiveness of treatments, using the most commonly used control intervention as a common comparator. The default settings of the R packages "BUGSnet" and "GeMTC" were used for the implementation of NMA [52,53]. For the response rate results, the effect was measured as an odds ratio (OR) with a 95% credible interval (Crl) using the binomial distribution assumption and logit link function. For the results of the eight nerve conduction velocity indices, the effects were analyzed as MD with 95% Crl using the normal likelihood model and identity link function. Markov chain Monte Carlo (MCMC) simulations were set up with a burn-in of 20,000 iterations and a total of 50,000 iterations, and every 10th value was extracted. Convergence was graphically assessed using trace and density plots. Node splitting was performed to assess the consistency of the response rate and a leverage plot was used to compare the DIC of the model based on the consistency assumption and the inconsistent model for the secondary outcome. A heat map with all feasible comparisons was constructed using the relative effect estimates from the NMA. We used a surface under the curve cumulative ranking probabilities (SUCRA) plot to demonstrate the ranking of treatments.

Network Pharmacology Analysis of the Synergistic Mechanism of the ACP against DPN
All bioactive ingredients in the ACP were screened and retrieved from the Traditional Chinese Medicine Systems Pharmacology (TCMSP; https://tcmsp-e.com/) analysis platform [54]. In this study, components with oral bioavailability (OB) ≥ 30% and drug-likeness (DL) index ≥ 0.18 were selected as candidate ingredients. The target information of active ingredients was standardized using the Uniprot database (http:// www.uniprot.org/) with the species filter "Homo sapiens". Using "diabetic peripheral neuropathy" as the keyword, data on DPN-related target genes were obtained from the GeneCards database (http://www.genecards.org). For targets in GeneCards, only those with a score ≥ 10 were screened [55]. Venn diagrams of consensus targets between the ACP and DPN were constructed using the Bioinformatics and Evolutionary Genomics website (https://bioinformatics.psb.ugent.be/webtools/Venn/). Using Cytoscape (v. 3.9.1; https://cytoscape.org/), a network of the components of the ACP and DPN targets was created to graphically depict the complex interactions between compounds and targets. The degree of each node is measured using a layout tool: the larger the node in the network, the higher the degree. The STRING protein analysis platform (v. 11.5; https://string-db.org/), together with the protein categorization "Homo sapiens", was used to import the interacting gene targets of the ACP and DPN [56]. Protein interaction network analysis was performed and Cytoscape software version 3.9.1 [57] was used to construct protein-protein interaction (PPI) network maps. Gene targets with a degree of centrality above the average value were selected as hub targets. Gene ontology (GO) functional analysis was used as the primary method to describe the functions of gene targets, including biological processes, cellular components, and molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to identify common targets of the ACP and DPN in the signaling pathways. Metascape (https://metascape.org/), an online tool for gene enrichment analysis, incorporates more than 40 functional annotation datasets [58]. Hub targets were uploaded to the Metascape platform for GO and KEGG analyses. The data selection criterion was set at p < 0.05.

Study Identification
Based on the search strategy, 903 potentially relevant articles were identified through electronic searches of 10 databases. After excluding 37 duplicates, 866 articles were retrieved. After screening titles and abstracts, 743 articles that met at least one of the exclusion criteria were excluded. The full texts of the remaining 123 studies were assessed, and 75 articles were excluded for the reasons listed in Figure 1. Finally, 48 eligible studies were included in our meta-analysis . The screening process is summarized in the PRISMA 2020 flow diagram ( Figure 1).
Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to identify common targets of the ACP and DPN in the signaling pathways. Metascape (https://metascape.org/), an online tool for gene enrichment analysis, incorporates more than 40 functional annotation datasets [58]. Hub targets were uploaded to the Metascape platform for GO and KEGG analyses. The data selection criterion was set at p < 0.05.

Study Identification
Based on the search strategy, 903 potentially relevant articles were identified through electronic searches of 10 databases. After excluding 37 duplicates, 866 articles were retrieved. After screening titles and abstracts, 743 articles that met at least one of the exclusion criteria were excluded. The full texts of the remaining 123 studies were assessed, and 75 articles were excluded for the reasons listed in Figure 1. Finally, 48 eligible studies were included in our meta-analysis . The screening process is summarized in the PRISMA 2020 flow diagram ( Figure 1).

Study Characteristics
The basic characteristics of the included studies are summarized in Table 1. The 48 trials included in this review were published between 2004 and 2021. A total of 4308 participants in the included studies were divided into experimental (n = 2175) and control groups (n = 2133), with sample sizes ranging from 29 to 202 participants. The average age of participants ranged from 38.7 to 69.8 years. The duration of neuropathy manifestations ranged from one month to >15 years. In 21 trials, the effects of EAHM monotherapy were compared with those of the comparator [59,61,64,65,70,75,76,79,81-83,86-

Risk of Bias
The methodological quality of the 48 included studies is summarized in Table 2. The risk of bias in studies was assessed using the RoB 2 tool [46]. All the included studies had a high risk of bias in one or more domains. According to the RoB 2 evaluation criteria, the "overall risk of bias" is also regarded as high if the risk of bias is assessed to be high even for one domain. The overall risk of bias for all studies included in this review was also considered high. Studies that were rated as having a "high" overall risk of bias frequently lacked information on the randomization method, and the absence of a preregistered protocol made it difficult to address concerns about selective outcome reporting. Additionally, as most studies lacked a blinded design, every variation in the intended intervention had a substantial risk of bias.

Pairwise Meta-Analysis
A pairwise meta-analysis was conducted for each intervention (EACP, ECCP, EAWP, and ECWP) to evaluate the effects on the response rate, motor nerve conduction velocity (MNCV), and sensory nerve conduction velocity (SNCV) compared to the control group.

Safety Assessment
Of the studies included in this review, 20 reported adverse events [59,61,66,67,70,[74][75][76]79,80,83,84,86,89,91,93,96,99,100,103]. Of these, 12 studies reported no adverse events in either the treatment or control group [61,67,[74][75][76]79,80,83,86,91,96,99]. The adverse events reported in eight trials were mostly digestive disorders such as anorexia, nausea, abdominal blotting, and diarrhea. Additionally, skin rash was observed in two trials and mild dizziness was reported in one trial [70,84,89]. No serious adverse events were reported in any of the included trials, and no significant differences were observed in the frequency or characteristics of adverse events between the EAHM intervention and CM control groups. The details of all adverse events reported in each trial are summarized in Table 1.

Sensitivity Analysis
More than 10 trials were included in the meta-analysis of the EAWP and CM on the response rate. Because severe heterogeneity was observed in this analysis, a sensitivity analysis of the leave-one-out method was performed, and one trial that significantly affected heterogeneity was identified [61]. However, this study did not show evident differences from other studies, and no separate effect on the overall effect size ( Figure 3A,B). No additional sensitivity analysis was performed for the other pairwise meta-analysis items because there were no reports of more than 10 trials per outcome. Of the studies included in this review, 20 reported adverse events [59,61,66,67,70,[74][75][76]79,80,83,84,86,89,91,93,96,99,100,103]. Of these, 12 studies reported no adverse events in either the treatment or control group [61,67,[74][75][76]79,80,83,86,91,96,99]. The adverse events reported in eight trials were mostly digestive disorders such as anorexia, nausea, abdominal blotting, and diarrhea. Additionally, skin rash was observed in two trials and mild dizziness was reported in one trial [70,84,89]. No serious adverse events were reported in any of the included trials, and no significant differences were observed in the frequency or characteristics of adverse events between the EAHM intervention and CM control groups. The details of all adverse events reported in each trial are summarized in Table 1.

Sensitivity Analysis
More than 10 trials were included in the meta-analysis of the EAWP and CM on the response rate. Because severe heterogeneity was observed in this analysis, a sensitivity analysis of the leave-one-out method was performed, and one trial that significantly affected heterogeneity was identified [61]. However, this study did not show evident differences from other studies, and no separate effect on the overall effect size ( Figure  3A,B). No additional sensitivity analysis was performed for the other pairwise metaanalysis items because there were no reports of more than 10 trials per outcome.

Publication Bias
A contour-enhanced funnel plot analysis was performed to explore publication bias through the response rate, which was the outcome of most of the included studies. Since the pattern in the funnel plot displayed asymmetry, publication bias was deemed possible ( Figure 4). This finding was further confirmed using Egger's test (t = 10.10, df = 39, p < 0.0001) and Begg's test (z = 4.23, p < 0.0001).

Publication Bias
A contour-enhanced funnel plot analysis was performed to explore publication bias through the response rate, which was the outcome of most of the included studies. Since the pattern in the funnel plot displayed asymmetry, publication bias was deemed possible ( Figure 4). This finding was further confirmed using Egger's test (t = 10.10, df = 39, p < 0.0001) and Begg's test (z = 4.23, p < 0.0001).

Quality of Evidence According to Outcome Measures
In the comparison between the EAHM interventions and CM controls, the overall quality of evidence according to all outcome measures ranged from very low to moderate. The results of the GRADE assessment are presented in Table 3 and Table S3. ⊕⊕ LOW a,c CM: conventional medicine; EACP: East Asian herbal medicine monotherapy containing the Astragali Radix-Cinnamomi Ramulus herb-pair; EAWP: East Asian herbal medicine monotherapy without the Astragali Radix-Cinnamomi Ramulus herb-pair; ECCP: East Asian herbal medicine and conventional medicine combined therapy containing the Astragali Radix-Cinnamomi Ramulus herb-pair; ECWP: East Asian herbal medicine and conventional medicine combined therapy without the Astragali Radix-Cinnamomi Ramulus herb-pair. GRADE working group grades of evidence. High quality(⊕⊕⊕⊕): further research is unlikely to change our confidence in estimating this effect. Moderate quality(⊕⊕⊕ ): further research is likely to have an important impact on our confidence in estimating the effect, and may change the estimate. Low quality(⊕⊕ ): further research is likely to impact our confidence in the estimate of the effect and is likely to change the estimate. Very low quality(⊕ ): very uncertain about the estimate. a: study design with some bias in randomized or distributed blind. b: 95% confidence interval passes 0 (MD and SMD) or 1 (RR and OR), and other interventions are not satisfied. c: confidence intervals are less overlapping, or the heterogeneity is high.

Network Meta-Analysis
NMA was performed for all 16 treatments, and the network relationships between the treatments for each outcome are shown in Figure 5. Detailed information is summarized in Table 4, including the number of interventions and networks for each outcome, the number of patients, whether the network is closed, and the number of direct comparisons.  and SMD) or 1 (RR and OR), and other interventions are not satisfied. c: confidence intervals are less overlapping, or the heterogeneity is high.

Network Meta-Analysis
NMA was performed for all 16 treatments, and the network relationships between the treatments for each outcome are shown in Figure 5. Detailed information is summarized in Table 4, including the number of interventions and networks for each outcome, the number of patients, whether the network is closed, and the number of direct comparisons,.

Response Rate
The SUCRA plot for the response rate with MCB as a comparator is shown in Figure 6A.  Figure 6B shows similar trends for all comparisons. EACP showed significantly better results than lifestyle modification (

Motor Nerve Outcomes: MMNCV, PMNCV, UMNCV, TMNCV
The SUCRA plot for the MMNCV with MCB as a comparator is shown in Figure 7A. The highest-ranked treatments were ECCP (SUCRA = 0.872), ECWP (SUCRA = 0.783), and EAWP (SUCRA = 0.656). The heat map in Figure 7B shows similar trends for all comparisons. ECCP showed significantly better results than MCB (MD 3.67; 95% CrI 1.17 to 6.14) and NMD (MD 5.48; 95% CrI 0.70 to 10.28). ECCP was the best-ranked intervention in the network.  The SUCRA plot for the PMNCV with MCB as a comparator is shown in Figure 7C.   Figure S12 shows similar trends for all comparisons. The EAWP was the best-ranked intervention in the network, but the difference was not statistically significant.
The SUCRA plot for USNCV with MCB as the comparator is shown in Figure 8C. The highest-ranked treatments were ECCP (SUCRA = 0.785), EACP (SUCRA = 0.771), and EAWP (SUCRA = 0.552). The heat map in Figure 8D shows similar trends for all comparisons. ECCP was the best-ranked intervention in the network but was not statistically significant.

Inconsistency Test
Regarding the response rate, as a result of node-splitting analysis of six interventions including multiple studies, no significant heterogeneity was observed in any comparison Additionally, for all studies related to response rate, no finding supporting heterogeneity was confirmed in the comparison of the posterior mean deviance between the consistency and inconsistency models ( Figure S17). In the case of secondary outcomes, DIC was compared using a leverage plot, and no significant inconsistency model DIC values were observed for any outcome that violated the consistency assumption ( Figure S18A-H).

Active Ingredients and Anti-DPN Gene Targets of the ACP
The TCMSP platform was screened using the absorption, distribution, metabolism, and excretion (ADME) criterion index of OB ≥ 30% and DL ≥ 0.18 to identify the active components in the ACP. A total of 27 active ingredients derived from the ACP were identified. Of these, 20 compounds occurred in Astragali Radix, and seven occurred in Cinnamomi Ramulus ( Table 5). The DrugBank database contains information on 364 component-target relationships (Supplementary Table S4), and the GeneCards database contains information on 1157 human target genes associated with DPN (Supplementary Table S5). After intersection mapping, 57 consensus genes were identified as potential therapeutic targets of the ACP against DPN (Figure 9).    identified. Of these, 20 compounds occurred in Astragali Radix, and seven occurred in Cinnamomi Ramulus ( Table 5). The DrugBank database contains information on 364 component-target relationships (Supplementary Table S4), and the GeneCards database contains information on 1157 human target genes associated with DPN (Supplementary  Table S5). After intersection mapping, 57 consensus genes were identified as potential therapeutic targets of the ACP against DPN (Figure 9).  Table S5). After intersection mapping, 57 consensus genes were identified as potential therapeutic targets of the ACP against DPN (Figure 9).

Network Analysis of the ACP and DPN Targets
The ACP component-DPN target network was mapped using Cytoscape software version 3.9.1. As shown in Figure 10, the network contained 78 nodes and 148 edges. The degree of a single target in the ACP-DPN network indicates the number of linked nodes. Network tools were analyzed to examine the network, and the degree of the active component was rated. Table 6 lists the top ten active ingredients according to degree, betweenness, and closeness centralities.

PPI Network Construction
Using the STRING 11.5 platform, we imported the common targets and constructed a PPI network, as shown in Figure 11A. One target (MT-ND6) was excluded from the PPI network as it did not interact with any other target. The PPI network of intersecting targets contained 56 nodes and 612 edges. Nodes that satisfied the average value of degree centrality (21.47) were retrieved through an additional examination of topological attributes, and 30 targets were eliminated during screening. Figure 11B shows the PPI network of the hub targets. Table 7 lists the top 27 hub targets based on their degree of centrality. On the other hand, every PPI pair analyzed on the STRING platform is assigned a score. This score does not indicate the strength or specificity of the PPI, but rather its reliability based on the available evidence. Calculated on a scale of 0 to 1, the closer the score is to 1, the more likely it is that the PPI is true. The interaction scores for all PPI pairs utilized in the study are presented in Table S6.  Using the STRING 11.5 platform, we imported the common targets and constructed a PPI network, as shown in Figure 11A. One target (MT-ND6) was excluded from the PPI network as it did not interact with any other target. The PPI network of intersecting targets contained 56 nodes and 612 edges. Nodes that satisfied the average value of degree centrality (21.47) were retrieved through an additional examination of topological attributes, and 30 targets were eliminated during screening. Figure 11B shows the PPI network of the hub targets. Table 7 lists the top 27 hub targets based on their degree of centrality. On the other hand, every PPI pair analyzed on the STRING platform is assigned a score. This score does not indicate the strength or specificity of the PPI, but rather its reliability based on the available evidence. Calculated on a scale of 0 to 1, the closer the score is to 1, the more likely it is that the PPI is true. The interaction scores for all PPI pairs utilized in the study are presented in Table S6.

Gene Ontology and KEGG Pathway Enrichment Analysis
The results of the GO and KEGG analyses of the top 27 hub targets are shown in Figure 12. A total of 510 biological processes (BP) were identified, including the cellular response to chemical stress, positive regulation of cell migration, response to lipopolysaccharide, positive regulation of protein phosphorylation, regulation of inflammatory response, regulation of smooth muscle cell proliferation, the mitogen-activated protein kinase signaling (MAPK) cascade, and regulation of epithelial cell migration ( Figure 12A). A total of 93 molecular functions were identified including cytokine activity, heme binding, MAP kinase activity, chromatin binding, metalloendopeptidase activity, fibronectin binding, integrin binding, carboxylic acid binding, kinase activator activity, and protease binding ( Figure 12B). A total of 33 cellular components were identified, including the membrane raft, vesicle lumen, endocytic vesicle, early endosome, external side of the plasma membrane, transcription regulator complex, extracellular matrix, and endoplasmic reticulum lumen ( Figure 12C). A total of 135 pathways were identified using KEGG pathway analysis ( Figure 12D). The results suggested that the mechanisms of the ACP were mainly linked to fluid shear stress, atherosclerosis, and the interleukin-17 (IL-17), MAPK, and vascular endothelial growth factor (VEGF) signaling pathways (Table S7).

Summary of the Findings
In our study, EAHM interventions were classified into four categories depending on the inclusion of the ACP and combination therapy with CM, and the comprehensive efficacy of EAHM interventions against DPN was compared with that of the CM control. EAHM showed considerably higher efficacy against DPN than the CM control, as de-

Summary of the Findings
In our study, EAHM interventions were classified into four categories depending on the inclusion of the ACP and combination therapy with CM, and the comprehensive efficacy of EAHM interventions against DPN was compared with that of the CM control. EAHM showed considerably higher efficacy against DPN than the CM control, as determined by the response rate, SNCV, and MNCV indices, regardless of the mode of usage. The EAHM formula containing ACP was ranked highest in NMA for each treatment in terms of response rate, MMNCV, PMNCV, MSNCV, and USNCV. As a result, the ACP appears to be a candidate combination that can significantly influence the therapeutic response and nerve damage recovery in DPN. Based on network pharmacology analysis, the aforementioned study predicted that 10 compounds, including quercetin, kaempferol, isorhamnetin, formononetin, and beta-sitosterol, would act on 27 targets.

Strengths and Limitations
This study has the following strengths: First, there are countless meta-analyses related to EAHM; however, to the best of our knowledge, this is the first network meta-analysis to investigate the synergistic effect of an herb-pair. The analysis performed in this study is expected to be useful in identifying the synergistic effects of EAHM through continuous improvements and developments in the future. Second, the mechanism of EAHM was reviewed at a deeper level using network pharmacology analysis in conjunction with NMA in clinical studies. Because the mechanism of action of EAHM is complex, detailed pharmacological information is often not discussed in clinical studies. Therefore, this study is valuable because it supports the efficacy hypothesis for DPN to be tested in EAHM clinical research. Third, the overall direction of this study was consistent with the proposal for determining candidate combinations for drug discovery. Meta-analysis is one of the most important clinical research methodologies; however, in the case of EAHM, personalized prescription is advantageous, and it is difficult to draw a firm conclusion about which material is valid owing to the heterogeneity between different EAHM formulae. The authors suggest that meta-analysis may be a useful tool for developing new drug candidates by scientifically validating the tacit knowledge associated with complex EAHM combinations.
Due to the following limitations, caution should be exercised when interpreting the results of this study: First, although the EAHM formula containing the ACP at various NMA endpoints occupied the highest rank, the results were not consistent in terms of all indicators. This is mainly because the interactions with herbs other than the ACP also affect the efficacy, and few studies have performed a stable-effect comparison between multiple treatments. However, the design of this study was based on the premise that the ACP is a combination with appropriate compatibility, and its synergistic effect is stronger than that for other herbal combinations. To overcome these limitations, additional clinical trials are required to conduct updated NMA. Second, the quality of the studies included in the NMA was generally low, and no RCTs employed a double-blind design. This is another limitation that can affect the results. As a follow-up to this review, the validation of the effect of the ACP may be firmly established with new clinical trials with an improved design in the future. Third, in this study, the mechanism was analyzed using network pharmacology. However, as the compounds and targets of ACP have not yet been fully identified, databasebased mechanism analysis based on data from previous studies revealed only predictive and not definitive mechanisms. Therefore, conclusions regarding the synergistic effects of the ACP and DPN can only be drawn through experimental studies. Prior to experimental testing, this study should be accepted to provide guidance.

Implications for Clinical Decision-Making
The significant difference between the effects of EAHM and the CM control, which was supported by the PMA data, is important because CM was used as a comparative treatment in most studies. Moreover, these results are encouraging because they are consistent with previous studies of similar design that investigated the effect of EAHM on DPN [31][32][33]38,107]. However, such a meta-analysis, which includes several types of EAHM formulae, has many limitations in its direct application to clinical decisionmaking, owing to strong heterogeneity due to differences in intervention composition and dose. Nevertheless, the consistent efficacy demonstrated by the findings of PMA in several previous studies and this review reinforces the idea that EAHM is a highly valuable candidate for drug discovery, at least for DPN treatment.
The EAHM formula containing the ACP occupied the highest rank among the multiple indicators included in the NMA target. Clinical evidence has established that EAHM formulae containing the ACP are useful for DPN, and the related mechanisms have been extensively explored [38,41,42,108]. Considering this and the fact that the ACP has long been used in combination with several EAHM prescriptions, the compatibility between the two components of the ACP is supported academically and historically. Moreover, both Astragali Radix and Cinnamomi Ramulus that make up the ACP have been shown to separately exert a wide range of pharmacological effects on systemic diseases, including the nervous, immune, endocrine, and cardiovascular systems, and are widely used medicinal plants [109][110][111][112]. Overall, EAHM formulae containing the ACP are considered superior for the treatment of DPN, and the development of a new drug for DPN using ACP or an EAHM combination containing the ACP as a candidate component seems valuable.

Implications for Drug Discovery
It is important to understand the herb-pair theory of EAHM outlined in the introduction to accurately predict the synergistic effects of herbal medicine combinations and apply it for drug discovery [22,[24][25][26][113][114][115]. EAHM is often used as a polyherbal mixture following established academic principles. The synergistic effects of these mixtures are expected to improve their efficacy while lowering the potential toxicity of the individual herbs. This is made feasible by the basic prescription premise of EAHM, which is "Gun-Shin-Jwa-Sa" (King-Retainer-Officer-Messenger in English) [27]. The places of "Gun" and "Shin" are given to herbs that have the strongest influence and in greater doses. In contrast, relatively smaller doses of herbs are considered at "Jwa" and "Sa" to reduce adverse effects or boost synergistic effects. Thus, a suitable herbal combination can exhibit amplified efficacy compared to a single herb [30,[116][117][118][119][120]. To establish these synergistic effects, an appropriate combination of EAHMs must be selected for drug development. Herb-pair theory is the most fundamental theory for compatibility [28,121,122]. This is extremely helpful as a research hypothesis for evaluating synergistic effects because it facilitates the development of an EAHM formula through the combination of two or three herbs. Therefore, in recent years, an increasing number of studies have used various methods to identify the synergistic mechanisms of potentially useful herb-pairs [123][124][125]. A previous study using a combination of network pharmacology and bioinformatics reported that Astragali Radix, which was also used in this study, could form a promising herbal pair for the treatment of DPN with Notoginseng Radix [126,127]. We combined network meta-analysis and network pharmacology analyses with reference to the latest studies to investigate the clinical effects and synergistic mechanisms of the ACP simultaneously. We found that DPN treatment using an EAHM involving the ACP is closely related to the IL-17 signaling pathway. The IL-17 cytokine family is primarily associated with acute and chronic inflammation. Accordingly, this pathway is considered a therapeutic target for chronic inflammatory diseases in humans, and blocking this pathway prevents the onset of type 1 diabetes in rodent models [128,129]. In addition, a recent cross-sectional study confirmed that the development of peripheral neuropathy in patients with type 2 diabetes was independently and positively associated with elevated IL-17 levels. This study suggests that IL-17 may have greater diagnostic value for DPN than other inflammatory cytokines [130]. The ACP is also involved in the regulation of the MAPK pathway. This is important because the MAPK cascade is a major factor in DPN pathogenesis. Recent studies have shown that nerve growth factors induced by high blood glucose levels promote an increase in MAPK levels, which contributes to an increase in the levels of inflammatory mediators that cause DPN, such as tumor necrosis factor (TNF) and IL-1. Increased levels of MAPKs are also involved in the pathogenesis of DPN via inflammatory cytokines via the activation of c-Jun/JNK. Therefore, the inhibitory effect of the ACP on this mechanism is significant because activation of MAPKs contributes to the overall progression of DPN [131]. Additionally, neuroinflammation and neurodegeneration are important pathologies in diabetic complications including DPN. Hyperglycemia-induced reactive metabolites damage the blood vessels and promote capillary thickening and endothelial proliferation. The resulting decreased oxygen supply and increased reactive oxygen species (ROS) synthesis further damage the neurons and induce vascular endothelial growth factor (VEGF) expression. Therefore, VEGF has been extensively studied as a primary singlemolecule target for the treatment of DPN, and our study predicted that the ACP could exert its therapeutic effect on DPN based on its action on this target [132]. Collectively, these potential predictive mechanisms and the fact that neuroinflammation is one of the major pathologies of DPN suggest that the ACP's mechanism of action is likely related to the inhibition of inflammation-induced neuronal degeneration. [10]. In this regard, the ACP may have neuroprotective effects similar to those of berberine, which served as a target in a rat model of diabetic neuropathy, resulting in better neuritin expression and micropathology [133,134].
The results of our study showed that ten active components, according to degree centrality, ensured the main effects of the ACP. Among these compounds, quercetin, kaempferol, isorhamnetin, formononetin, and beta-sitosterol are thought to exert synergistic effects [135,136]. Quercetin is a promising candidate compound for a multitargeted approach to the complications of type 2 diabetes and has been reported to reduce oxidative stress, protect beta cells, and stimulate glucose uptake in muscle cells via the AMPK pathway [137]. In addition, it prevented diabetic complications by alleviating oxidative stress-induced apoptosis in a rat model of type 1 diabetes [138]. Kaempferol inhibits hyperglycemia-induced RhoA activation and diabetic kidney disease by reducing oxidative stress and proinflammatory cytokine levels [139]. Isorhamnetin is known for its various physiological activities, including neuroprotective, anti-inflammatory, antioxidative, and immunomodulatory effects [140,141]. DPN-related mechanisms have also been reported to prevent hyperglycemia by promoting glucose uptake by skeletal muscle cells and inhibiting insulin resistance [142,143]. Formononetin suppresses neuronal damage by controlling hyperglycemia in a rat model of diabetic neuropathy, improves nerve conduction velocity, and elicits synergistic effects by reducing thermal hyperalgesia and mechanical allodynia [144]. Finally, beta-sitosterol has been reported to have neuroprotective and antinociceptive effects in an animal model of diabetic neuropathic pain, based on insulin secretion promotion, alpha-glucosidase inhibition, blood sugar suppression, and antioxidant action [145]. In summary, several physiologically active ingredients present in the ACP may have synergistic effects on the prevention of nerve damage, repair of damaged nerves, and inhibition of DPN progression through an antidiabetic action via multiple pathways. This finding is consistent with the results of NMA in clinical trials. Therefore, the ACP is a promising candidate combination and its synergistic effects must be verified through subsequent experimental studies.

Conclusions
EAHM may promote therapeutic efficacy in the management of DPN; when combined with CM, it can treat DPN significantly more effectively than CM alone. EAHM formulae containing the ACP may be more suitable than other EAHM formulae for improving NCV and the treatment response rate to DPN therapy, as determined by a network meta-analysis. The ACP has also been shown to cure DPN synergistically via multiple pathways and may work primarily through the IL-17 and MAPK signaling pathways to alter the pathophysiology of chronic diabetes mellitus in peripheral nerves. These observations suggest that ACP could be used to treat pain, paresthesia, neuroinflammation, and neurodegeneration in patients with DPN.
The key ingredients in the ACP, including quercetin, kaempferol, isorhamnetin, formononetin, and beta-sitosterol, might have synergistic effects in neuroprotection, antiinflammation, antioxidation, immunomodulation, and antinociception. These results suggest that ACP is a useful candidate for the treatment of DPN and should be studied further. However, the findings of this study should be verified through clinical trials and experimental studies. Despite these limitations, this study is valuable for proposing research hypotheses for candidate natural therapeutics based on herbal pairs and their synergistic effects.
Finally, the above study exploring the synergistic effects of herbal medicine combinations is expected to verify the efficacy of licensed herbal medicines and classical EAHM prescriptions consisting of multiple herbs based on a traditional holistic perspective. Currently, the standardization of herbal formulations mainly depends on the content of a single indicator component; however, this information is insufficient to support efficacy. Therefore, an advanced test method to clarify the multilayered indications of herbal medicines is essential. To clarify the indications for complex herbal medicine prescriptions beyond the mechanisms and effects of individual herbal medicines, it is necessary to continuously and multidimensionally verify the synergistic effects explored in this study.  Author Contributions: H.-G.J.: conceptualization, methodology, software, validation, formal analysis, investigation, resources, data curation, writing-original draft preparation, writing-review and editing, visualization, and project administration; E.B.: conceptualization, methodology, validation, writing-original draft preparation, and writing-review and editing; D.L.: conceptualization, methodology, validation, formal analysis, investigation, writing-review and editing, supervision, project administration, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The data in this study were all utilized from public databases and published studies and had unrestricted access.